New Antibodies Could Help Researchers Track TDP-43 Changes in ALS and Frontotemporal Dementia
Researchers developed highly sensitive antibodies that detect HDGFL2-CE, an abnormal protein associated with TDP-43 dysfunction, creating new tools for ALS and frontotemporal dementia research.
Key Takeaways
- Researchers from the University of Miami Miller School of Medicine developed more sensitive antibodies that detect HDGFL2-CE, an abnormal protein associated with TDP-43 dysfunction.
- The antibodies detected smaller amounts of HDGFL2-CE than the team’s first-generation antibody and distinguished the abnormal protein from normal HDGFL2.
- The tools could support future biomarker research for conditions including ALS and frontotemporal dementia.
Researchers from the Neuroscience Institute at the University of Miami Miller School of Medicine and collaborating institutions have developed more sensitive tools to study a biological process involved in amyotrophic lateral sclerosis, or ALS, and frontotemporal dementia.
The new antibodies detect HDGFL2-CE, an abnormal protein associated with dysfunction of TDP-43, which helps nerve cells process genetic instructions. The findings, published in Cell Reports Medicine, strengthen the case for studying HDGFL2-CE as a potential marker of TDP-43-related disease.
Miller School members of the research team included:
- Leonard Petrucelli, Ph.D., founding director of the Miller School’s Neuroscience Institute
- Mercedes Prudencio, Ph.D., associate professor of neurology and biochemistry and molecular biology
- Yongjie Zhang, Ph.D., associate professor of biochemistry and molecular biology
- Tania Gendron, Ph.D., associate professor of neurology
- Vidhya Bharathi Maheswari Jawahar, Ph.D., a Miller School researcher
- Janie Citrano, a Miller School graduate student
What Goes Wrong With TDP-43?
TDP-43 is a protein that normally works inside the cell nucleus, where it helps process RNA. RNA carries the instructions cells used to make proteins.
In most people with ALS and many people with frontotemporal lobar degeneration involving TDP-43, the protein moves out of the nucleus and accumulates in cytosol in nerve cells, known as TDP-43 pathology. This disruption can cause errors in RNA splicing, the editing process cells used to assemble genetic instructions correctly.
One type of error causes cells to retain unwanted pieces of genetic material known as cryptic exons. These pieces are normally excluded from mature RNA. In some cases, their inclusion causes cells to insert new protein fragments into normal proteins, creating abnormal proteins that are not normally found in the body.
One of those proteins is HDGFL2-CE.
Why Were New Antibodies Needed?
Previous studies had detected HDGFL2-CE in brain tissue, cerebrospinal fluid and blood from people with TDP-43-related disorders. Those findings suggested that the protein could become a biomarker of disease activity.
Existing antibodies, however, were not sensitive enough to fully investigate the protein’s biological and potential clinical significance. To improve detection, the team developed polyclonal and monoclonal antibodies designed to recognize the abnormal portion of HDGFL2-CE.
“One of the major challenges in ALS and frontotemporal dementia research is that we currently lack a reliable biomarker that can identify TDP-43 pathology in living patients,” said Dr. Zhang. “HDGFL2-CE is particularly promising because it reflects a molecular consequence of TDP-43 pathology and associated dysfunction and, therefore, has the potential to serve as a biomarker of this underlying disease process.”
Researchers tested the antibodies using laboratory-made proteins, human cell models and donated brain tissue to confirm that they detected the intended target.
The new LP087 and LP088 antibodies detected much smaller amounts of HDGFL2-CE than the group’s first-generation antibody. In laboratory experiments, the assay detected concentrations as low as approximately 0.041 picograms per microliter. They also distinguished the abnormal protein from normal HDGFL2.
What Did Researchers Find in Brain Tissue?
The researchers used the improved assay to analyze frontal-cortex tissue from 450 people across four groups:
- 79 cognitively normal controls
- 222 people with frontotemporal lobar degeneration involving TDP-43
- 89 people with the same condition accompanied by motor neuron disease
- 60 people with motor neuron disease
HDGFL2-CE levels were significantly higher in the two frontotemporal lobar degeneration groups than in the control group. Higher levels of HDGFL2-CE were also associated with greater levels of phosphorylated TDP-43, an abnormal form of the protein that accumulates in affected nerve cells.
The marker was associated with clinical measures of disease burden, as well. People with higher HDGFL2-CE levels tended to develop symptoms at younger ages and had shorter survival after symptoms began. These relationships are associations and do not establish that HDGFL2-CE causes earlier disease onset or faster progression.
Microscopy provided additional evidence connecting HDGFL2-CE with TDP-43 dysfunction. The abnormal protein accumulated in neurons that contained TDP-43 inclusions and had lost TDP-43 from the nucleus. Healthy control tissue showed little or no HDGFL2-CE staining.
How TDP-43 Dysfunction Can Produce HDGFL2-CE
- Normal function: TDP-43 works in the cell nucleus and helps process RNA.
- Disruption: TDP-43 can leave the nucleus and accumulate elsewhere in the nerve cell.
- RNA-splicing error: Cells may retain unwanted genetic sequences called cryptic exons.
- Abnormal protein: One of the resulting proteins is HDGFL2-CE.
- Research detection: The new antibodies allow researchers to detect much smaller amounts of HDGFL2-CE.
Could HDGFL2-CE Become a Biomarker?
The researchers also tested whether the immunoassay could work reliably in cerebrospinal fluid, the liquid surrounding the brain and spinal cord. The assay successfully detected small amounts of HDGFL2-CE added to CSF samples, suggesting that it may be suitable for measuring HDGFL2-CE in patient biofluids in future studies.
The new assay has not yet been used to measure naturally occurring HDGFL2-CE in clinical cerebrospinal fluid cohorts. Additional validation is needed before HDGFL2-CE could be used in clinical care. The overlap between disease and control groups also suggests that it is unlikely to work as a standalone diagnostic. It may ultimately be more useful as part of a broader biomarker panel interpreted alongside clinical and pathological information.
What Comes Next?
The antibody toolkit gives researchers multiple ways to recognize different regions of the abnormal HDGFL2-CE protein. These tools could support future biomarker studies and help investigators examine how closely HDGFL2-CE tracks TDP-43 dysfunction in cells, tissue and patient biofluids.
“One of our next priorities is to develop ultrasensitive immunoassays so that we can reliably detect HDGFL2-CE in patient biofluids, such as cerebrospinal fluid and blood,” said Dr. Zhang. “We are currently developing antibodies against other cryptic exon-derived proteins, with the goal of building a multiplex biomarker panel that better captures TDP-43 pathology and associated dysfunction.”
The work also raises an important question. Is HDGFL2-CE simply a measurable result of TDP-43 dysfunction or does the abnormal protein contribute to nerve cell damage?
“These tools will allow us to ask broader biological questions about what happens when these abnormal proteins are produced,” said Dr. Petrucelli.
More from the Miller School of Medicine
A TMEM106B variant is linked to TDP-43 pathology in Alzheimer’s disease, offering clues to why cognitive decline differs among patients.
Two Miller School Safenowitz Fellowship-supported researchers are studying how immune responses may contribute to ALS.
Researchers identified an abnormal UNC13A RNA signature linked to faster cognitive decline and greater impairment in Alzheimer’s disease.
Dr. Leonard Petrucelli leads two major grants advancing collaborative Parkinson’s disease research at the Miller School.
Tags: Aging Research, ALS, amyotrophic lateral sclerosis, biochemistry and molecular biology, cognitive decline, Department of Biochemistry and Molecular Biology, Dr. Leonard Petrucelli, Dr. Mercedes Prudencio, Dr. Tania Gendron, Dr. Yongjie Zhang, Frontotemporal dementia, memory disorders, Neuroscience Institute